A. Field of the Invention
The present invention relates to methodology and associated genetic constructs for the suppression of oncogene-mediated, transformation, tumorigenesis and metastasis. In particular, this invention relates to the suppression of oncogenesis that is mediated by the HER-2/c-erb B-2/neu oncogene, an oncogene which has been correlated with a poor prognosis of breast and ovarian carcinoma in humans.
B. Background of the Related Art
During the last decade, a number of human malignancies have been discovered to be correlated with the presence and expression of xe2x80x9concogenesxe2x80x9d in the human genome. More than twenty different oncogenes have now been implicated in tumorigenesis, and are thought to play a direct role in human cancer (Weinberg, 1985). Many of these oncogenes apparently evolve through mutagenesis of a normal cellular counterpart, termed a xe2x80x9cproto-oncogenexe2x80x9d, which leads to either an altered expression or activity of the expression product. There is considerable data linking proto-oncogenes to cell growth, including their expression in response to certain proliferation signals (see, e.g., Campisi et al., 1983) and expression during embryonic development (Muller et al., 1982). Moreover, a number of the proto-oncogenes are related to either a growth factor or a growth factor receptor.
The c-erbB gene encodes the epidermal growth factor receptor (EGFr) and is highly homologous to the transforming gene of the avian erythroblastosis virus (Downward et al., 1984). The c-erbB gene is a member of the tyrosine-specific protein kinase family to which many proto-oncogenes belong. The c-erbB gene has recently been found to be similar, but distinct from, an oncogene referred to variously as c-erbB-2, HBER-2 or neu oncogene (referred to herein simply as the neu oncogene), now known to be intimately involved in the pathogenesis of cancers of the human female breast and genital tract.
The neu oncogene, which encodes a p185 tumor antigen, was first identified in transfection studies in which NIH 3T3 cells were transfected with DNA from chemically induced rat neuroglioblastomas (Shih et al., 1981). The p1 85 protein has an extracellular, transmembrane, and intracellular domain, and therefore has a structure consistent with that of a growth factor receptor (Schechter et al, 1984). The human neu gene was first isolated due to its homology with v-erbB and EGF-r probes (Senba et al., 1985).
Molecular cloning of the transforming neu oncogene and its normal cellular counterpart, the neu proto-oncogene, indicated that activation of the neu oncogene was due to a single point mutation resulting from one amino acid change in the transmembrane domain of the neu encoded p185 protein (Bargmann et al., 1986; Hung et al., 1989).
The neu oncogene is of particular importance to medical science because its presence is correlated with the incidence of cancers of the human breast and female genital tract. Moreover, amplification/overexpression of this gene has been directly correlated with relapse and survival in human breast cancer (Slamon et al., 1987). Therefore, it is an extremely important goal of medical science to evolve information regarding the neu oncogene, particularly information that could be applied to reversing or suppressing the oncogenic progression that seems to be elicited by the presence or activation of this gene. Unfortunately, little has been previously known about the manner in which one may proceed to suppress the oncogenic phenotype associated with the presence of oncogenes such as the neu oncogene.
An extensive body of research exists to support the involvement of a multistep process in the conversion of normal cells to the tumorigenic phenotype (see, e.g., Land et al., 1983). Molecular models supporting this hypothesis were first provided by studies on two DNA tumor viruses, adenovirus and polyomavirus. In the case of adenovirus, it was found that transformation of primary cells required the expression of both the early region 1A (E1A) and 1B (E1B) genes (Houweling et al., 1980). It was later found that the E1A gene products could cooperate with middle T antigen or with activated H-ras gene to transform primary cells (Ruley, 1985). These observations suggested that the involvement of multiple functions in the transformation process, and that various oncogenes may express similar functions on a cellular level.
The adenovirus E1A gene codes for several related proteins to which a number of interesting properties have been attributed. In addition to its ability to complement a second oncogene in transformation, a closely related function allows E1A to immortalize primary cells (Ruley, 1985). For example, introduction of E1A gene products into primary cells has been shown to provide these cells with an unlimited proliferative capacity when cultured in the presence of serum.
Another interesting action of E1A function is so-called xe2x80x9ctrans-activationxe2x80x9d, wherein E1A gene products stimulate transcription from a variety of viral and cellular promoters, including the adenovirus early and major late promoter. However, trans-activation is not universal for all promoters. In some instances, E1A causes a decrease in transcription from cellular promoters that are linked to enhancer elements (Haley et al., 1984). Recently, it has been shown that exogenously added E1A gene can reduce the metastatic potential of ras-transformed rat embryo fibroblast cells by activating the cellular NM23 gene that is associated with a lower metastatic potential (Pozzatti et al., 1988; Wallich et al., 1985).
The E1A gene products are referred to as the 13S and 12S products, in reference to the sedimentation value of two mRNAs produced by the gene. These two mRNAs arise through differential splicing of a common precursor, and code for related proteins of 289 and 243 amino acids, respectively. The proteins differ internally by 46 amino acids that are unique to the 13S protein. A number of E1A protein species can be resolved by PAGE analysis, and presumably arise as a result of extensive post-translational modification of the primary translation products (Harlow et al., 1985).
Another viral oncoprotein, the SV 40 large T antigen (LT) shares structural and functional homology to E1A and c-myc (Figge et al., 1988). LT, E1A and c-myc have transforming domains which share amino acid sequence homology and similar secondary structure (Figge et al., 1988). All three proteins complex with the tumor suppressor, retinoblastoma gene product (Rb) (Whyte et al., 1988, DeCaprio et al., 1988, Rustgi et al., 1991), and the Rb binding domains of LT and E1A coincide with their transforming domains. Based on this similarity, it has been thought that LT and E1A transform cells by binding cellular Rb and abrogating its tumor suppressor function. LT, E1A and c-myc are also grouped as immortalization oncogenes as determined by the oncogene cooperation assay using rat embryo fibroblasts (Weinberg, 1985).
In spite of the similarity between the Rb binding domains of LT and E1A, the two proteins differ substantially in other regards. In fact, there is apparently only a short equivalent stretch of acidic amino acids (Figge et al., 1988). This stretch lies between amino acids 106-114 in LT and amino acids 121-139 in E1A. The large T antigen is encoded by the simian virus 40, a member of the polyoma virus family. In contrast, E1A is encoded by adenovirus 5 virus, which is a member of the adenovirus family. LT is 708 amino acids long, while E1A is substantially shorter at 298 amino acids. LT has been observed to bind directly to certain DNA sequences, however, E1A has not. LT binds with the tumor suppressors Rb and also with p53. E1A complexes with Rb but not with p53. E1A has been shown to induce apoptosis in cells, this has not been demonstrated for LT.
Further, LT is an apparent anomaly in the scheme of oncogenic classification. Oncogenes are typically classified as being cytoplasmic or nuclear oncogenes. However, LT, through the actions of a single protein, is able to introduce xe2x80x9cnuclearxe2x80x9d characteristics such as immortalization and xe2x80x9ccytoplasmicxe2x80x9d characteristics such as anchorage independence in cells (Weinberg, 1985). LT antigen can be found in both the nucleus and at the plasma membrane, and mutations that inhibit the transport of LT into the nucleus appear to reduce its immortalizing ability while leaving intact its effect on anchorage independence and its ability to transform already immortalized cells. Consequently, this oncogene is considered to be a member of both the nuclear and cytoplasmic oncogenic classes, since it sends its gene product to do work at two distinct cellular sites (Weinberg, 1985). In contrast, E1A is known as a nuclear oncogene only.
Despite advances in identifying certain components which contribute to the development of malignancies, it is clear that the art still lacks effective means of suppressing carcinogenesis. For example, there is as yet no particularly successful way of suppressing neu oncogene activation or the development of various cancers, such as those of the breast and genital tract, which are associated with this molecular event.
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing methods for the suppression of neu-mediated oncogenesis. Certain aspects of the present invention relate to the inventors"" surprising discovery that, in contrast to previous characterizations of the E1A gene and the LT gene as being involved in promoting transformation, the E1A and LT gene products can actually serve to suppress not only the expression of the neu oncogene, but suppress the oncogenic phenotype which accompanies neu oncogene activation. Furthermore these gene products sensitize cancer cells to chemotherapeutic agents. It is proposed that this exciting discovery opens the door to novel approaches to the treatment of neu oncogene-mediated cancers, as well as an improved understanding of the regulation of this oncogene in particular and the oncogenic phenotype in general.
The present invention thus arises out of the inventors"" surprising discovery that products of the adenovirus E1A gene, a gene that is itself known to serve as an oncogene, can be effectively employed to suppress the transforming capability of the neu oncogene and sensitize chemoresistant cancer cells to chemotherapeutic regimens. Accordingly, the invention can be characterized in a general sense as relating to a method of treating neu oncogene-mediated transformation of a cell, which method includes introducing an E1A gene product into such a cell in a manner that is effective to suppress an oncogenic phenotype, as indicated by a reduction in transforming, tumorigenic or metastatic potential of the cell. Further the introduction of the E1A gene product into the cell sensitizes it to conventional chemotherapeutic agents. Hence gene therapy is used in conjunction with chemotherapy to effectively kill the cancer.
The invention also arises out of the inventors"" surprising showing that introduction of LT antigen into cells leads to a significant decrease in the expression of neu encoded p1 85 and also sensitizes neu-overexpressing cells to chemotherapeutic agents. LT, like E1A and c-myc, represses the upstream regulatory sequences of neu. However, LT represses a different region of the neu regulatory sequences compared to E1A and c-myc, suggesting LT affects neu expression through a different pathway.
Thus the present invention, in a general and overall sense, concerns methods of inhibiting oncogene-mediated transformation of a cell and sensitizing the cell to chemotherapeutic agents using gene products. These methods involve contacting the cell with a neu-suppressing gene product and a chemotherapeutic drug in amounts effective to inhibit the transformed phenotype.
The objects of the invention may be achieved by introduction of E1A gene products or LT intracellularly in any convenient manner, including, for example, virus mediated gene transfer, DNA transfection via calcium phosphate or liposome methods, and even direct introduction of gene products by microinjection. It is proposed that methods such as these will work adequately, e.g., where one is seeking to study neu oncogene suppression. However, where a treatment regimen is contemplated it will likely be necessary to introduce the selected E1A gene product or LT by intracellular introduction of a DNA segment which encodes the particular domain of the E1A protein or LT that is required for repression of neu.
In any event, since the E1A gene products have been extensively characterized, and the gene itself has been cloned (see, e.g., Berk et al., 1978), the starting materials, i.e., the E1A products and gene, are readily available to those of skill in the art who desire to practice the invention.
LT is also characterized and the gene has been cloned. The entire SV40 nucleotide sequence is disclosed in the book Molecular Biology of Tumor Viruses, Part 2, 2d. ed., Tooze, J., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1981), Appendix A, pgs. 799-813. In addition to the genomic sequence, Molecular Biology of Tumor Viruses contains a map of SV40 landmarks including the location of the large T antigen within the SV40 genome [pg. 813]. The references Fiers et al., 1978 and Reddy et al., 1978 also report the genetic sequences of SV40. The amino acid sequence of LT can be found in Molecular Biology of Tumor Viruses, pgs. 854 and 857-861. Various mutant of native LT have been described. For example, Kalderon et al. (1984) describe many LT mutations, which were the result of deletion and point mutations of the native LT gene. The relevant amino acid sequences of each LT mutant reported in Kalderon et al. are contained in Table 2 of that reference. By combining the information in Kalderon et al. (1984) with the sequence information for native LT contained in Molecular Biology of Tumor Viruses, the sequence for any of these mutants can be determined. All of the genomic and amino acid sequences of native LT and LT mutants contained in the references cited in this paragraph are incorporated by reference in this specification.
Some embodiments of the invention involve methods of inhibiting oncogene-mediated transformation of a cell. Generally, these methods comprise the step of contacting the cell with an oncogenic phenotype suppressing gene product and a chemotherapeutic drug in amounts effective to inhibit the transformed phenotype. In a preferred embodiment, the oncogene-mediated transformation being inhibited will be neu oncogene-mediated transformation. Also, preferably, the embodiments in which transformation is to be inhibited will comprises a tyrosine specific protein kinase encoded by neu. Of course, the invention also applies to methods of inhibiting other oncogene-mediated transformation events, such as transformation by ras, src, yes, fps, fes, abl, ros, fgr, erbB, fms, mos, raf etc.
Embodiments of the present invention involve chemotherapeutic agents. These are compounds that exhibit some form of anti-cancer activity. In some preferred embodiments, the chemotherapeutic drug is an alkylating agent, plant alkaloid, antibiotic, or antineoplastic agent. In those embodiments of the invention where the chemotherapeutic is an alkylating agent, the alkylating agent may be, for example, mechlorethamine, cyclophosphamide, ifosfamide chlorambucil, melphalan, busulfan, thiotepa, carmustine, lomustine, and/or streptozocin. In those embodiments where the chemotherapeutic agent comprises a plant alkaloid, the plant alkaloid is, for example, vincristine, vinblastine or taxol. In a preferred embodiment, the plant alkaloid is taxol. In those embodiments of the invention where the chemotherapeutic agent is an antibiotic, the antibiotic may be, for example, dactinomycin, daunorubicin, idarubicin, bleomycin mitomycin or doxorubicin. In most preferred embodiments the antibiotic is doxorubicin. In other embodiments where the chemotherapeutic agent comprises an antineoplastic, the preferred antineoplastic is, for example, cisplatin, VP16 and TNF.
In certain embodiments of the invention, the E1A or LT is administered to the cell prior to the administration of the chemotherapeutic agent. In other aspects of the invention, the chemotherapeutic agent is administered to the cell prior to administration of the E1A or LT. Alternatively the E1A or LT and the chemotherapeutic drug are administered simultaneously.
In some embodiments of the invention, the cell is located within an animal and effective amounts of the E1A or LT and the chemotherapeutic drug are administered to the animal. In certain embodiments of the invention, the chemotherapeutic drug and the E1A or LT are suitably dispersed in a pharmacologically acceptable formulation. In certain preferred embodiments where the cell is an animal cell, the animal cell is a human cell. In other preferred embodiments the cells is a lung, cancer cell, ovarian cancer cell, or a breast cancer cell.
In some embodiments of the present invention the cell is contacted with a single composition comprising the E1A or LT in combination with a chemotherapeutic agent. In such cases, the composition may be suitably dispersed in a pharmacologically acceptable formulation.
The invention contemplates embodiments comprising sensitizing a cancer cell to a chemotherapeutic drug. These embodiments comprise exposing the cell with an effective amount of the E1A or LT. In some such embodiments inhibition of neu-mediated cancer is accomplished by administrating an effective combination of the E1A or LT and chemotherapeutic drug to an animal having or suspected of having cancer in an effective amount to inhibit the cancer. In embodiments where the composition is administered to an animal, the animal is typically a mammal. In such cases, the invention will be of particular use in the treatment and prevention of neu-mediated transformation in humans
Certain embodiments of the present invention comprise injecting a therapeutically effective amount of the E1A or LT into an animal and contacting the animal with a chemotherapeutic drug. In certain embodiments of the invention the cancer site is contacted with a chemotherapeutic drug by administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic drug wherein the chemotherapeutic drug is for example cisplatin, doxorubicin, VP16, taxol or TNF.
The inventors have also enabled the production of pharmaceutical compositions comprising an E1A or LT and a chemotherapeutic drug in a pharmacological carrier. Those of skill will understand the nature of such pharmacological carriers based on the teachings of this specification and the current knowledge in the art. The pharmaceutical compositions of the invention may contain any of the E1A or LT and chemotherapeutic drugs mentioned above or elsewhere in this specification, or know to those of skill in the art. They may also contain emodin and/or an emodin like compound. In a preferred pharmaceutical composition the chemotherapeutic drug is cisplatin, doxorubicin, etoposide, taxol or TNF. In some preferred embodiments, the neu-suppressing gene product is E1A. In some preferred embodiments the neu-suppressing gene product is LT.
The invention also encompasses pharmaceutical combinations comprising an a neu-suppressing gene product and a chemotherapeutic drug. In certain preferred combinations, the neu-suppressing gene product is E1A. In certain other preferred combinations, the neu-suppressing gene product is LT. The chemotherapeutic drug may be any that is listed elsewhere in this specification or known to those of skill in the art at the present or in the future. Exemplary chemotherapeutic drugs for us in the pharmaceutical combinations of the present invention are cisplatin, doxorubicin, etoposide, emodin and or emodin like compounds, taxol and TNF. In certain embodiments of the invention the pharmaceutical combination may contain the E1A or LT and the chemotherapeutic drug within the same pharmaceutical composition. In other embodiments, the pharmaceutical combinations will comprise separate pharmaceutical compositions for each of the E1A or LT and the chemotherapeutic drug. These separate compositions may be combined internal to or external to a body to create the pharmaceutical combination.
Other embodiments of the invention include therapeutic kits comprising in suitable container, a pharmaceutical formulation of an the E1A or LT preparation, a pharmaceutical formulation of a chemotherapeutic drug, and/or a pharmaceutical formulation comprising both the E1A or LT and a chemotherapeutic drug. Emodin and/or emodin like compounds may also be present in the kit either in combination with the gene products, chemotherapeutic agent, gene products and chemotherapeutic agent or indeed in a separate formulation. The kit may also contain instructions on how to administer the pharmaceutical formulation or formulations of the kit to an animal either alone, or in combination with formulations that one may obtain separately from the kit. The kit may also comprise instructions that explain how to use the kit but are provided separately from the container of the kit. The kit may comprise the E1A or LT, emodin and/or emodin like compound, and chemotherapeutic drug to be present within a single container or alternatively the kit could comprise the E1A or LT and/or emodin and the chemotherapeutic drug are present within distinct containers.
Some embodiments of the present invention relate to a method of sensitization of a cell to an anticancer drug, comprising contacting the cell with the E1A or LT. These gene products are well-described in this specification. In preferred embodiments the oncogene-mediated transformation is neu oncogene-mediated transformation. Also, preferably, the embodiments in which transformation is to be inhibited will comprises a tyrosine specific protein kinase encoded by neu. The invention also contemplates pharmaceutical compositions, and kits comprising the E1A or LT to suppress neu-mediated transformation. Of course, the invention also applies to methods of inhibiting or suppressing other oncogene-mediated transformation events, such as transformation by ras, src, yes, fps, fes, abl, ros, fgr, erbB, fms, mos, raf.
Emodin-like tyrosine kinase inhibitors of the invention are those compounds that exhibit similar characteristics to those of emodin with regard to tyrosine kinase inhibition and the inhibition of neu-mediated transformation. Of course the invention is not limited to the use of these inhibitors and other inhibitors that possess the structural and/or functional properties of emodin may be used. In some preferred embodiments, the emodin-like tyrosine kinase inhibitor is an anthraquinone-like tyrosine kinase inhibitor. The emodin-like tyrosine kinase inhibitor may be, for example, emodin, emodin-8-O-D-glucoside, chrysophanic acid, gluco-chrysophanic acid, physcion, or physcion-8-O-D-glucoside. In the most preferred embodiment the neu tyrosine kinase inhibitor is emodin.
Other embodiments of the present invention relate to a method of inhibiting oncogene-mediated transformation of a cell, comprising contacting the cell with the E1A or LT, further contacting the cell with emodin and/or an emodin-like compound and further still contacting the cell with the chemotherapeutic agent. The cell may be contacted with the gene product, the emodin and/or emodin like compound and the chemotherapeutic agent successively in any order. Alternatively the cell is contacted with a combination of gene product and emodin, gene product and chemotherapeutic drug, emodin and chemotherapeutic drug followed or preceded by treatment of the third agent. In yet another embodiment it is possible to contact the cell with the gene product, emodin and/or emodin like compound and chemotherapeutic agent concurrently with each other.
In some embodiments of this invention an a cell is contacted with between about 0.5 mg/kg total weight and 500 mg/kg total weight of the emodin-like tyrosine kinase inhibitor. Other embodiments wherein the cell is contacted with between about 0.5 mg/kg total weight and 500 mg/kg total weight of emodin. In still other embodiments the cell is contacted with between about 0.5 mg/kg total weight and 500 mg/kg total weight of an emodin-like tyrosine kinase inhibitor. Total weight may be defined as the total weight of the cell or cells in culture, or the body weight of an animal, including a human.
The present invention also contemplates compositions comprising a liposomal complex. This liposomal complex will comprise a lipid component and a DNA segment encoding a neu-suppressing gene. The neu-suppressing gene employed in the liposomal complex can be, for example, an LT gene or an E1A gene. Liposomal complexes comprising LT mutants may have certain advantages. These advantages may be particularly distinct when the LT gene encodes non-transforming LT mutant, such as K1. An E1A gene encoding either the E1A 12S or E1A 13S gene product, or both, may be complexed with a lipid to form the liposomal complex.
The lipid employed to make the liposomal complex can be any of the above-discussed lipids. In particular, DOTMA, DOPE, and/or DC-Chol may form all or part of the liposomal complex. The inventors have had particular success with complexes comprising DC-Chol. In a preferred embodiment, the lipid will comprise DC-Chol and DOPE. While any ratio of DC-Chol to DOPE is anticipated to have utility, it is anticipated that those comprising a ratio of DC-Chol:DOPE between 1:20 and 20:1 will be particularly advantageous. The inventors have found that liposomes prepared from a ratio of DC-Chol:DOPE of about 1:10 to about 1:5 have been useful in the studies they have performed. In most studies, the inventors have used a ratio of 1.2 xcexcmol DC-Chol:8.0 xcexcmol DOPE.
The present invention also comprises kits for the introduction of a neu-suppressing gene product into a cell comprising a neu-suppressing DNA/liposome complex.